COAS Biology 1 Teacher ResourcesOriginal material © Cambridge University Press 20081
9 Practical 1
Following the course of an enzyme-catalysed reaction using amylase
Safety
The normal safety precautions associated with the use of chemicals apply.
Apparatus and materials
•colorimeter and cuvettes•water bath set at 25°C
•250cm3 beaker
•25cm3 beaker
•two plastic beakers
•100cm3 measuring cylinder
•20cm3 measuring cylinder
•1cm3 syringe
•two 5cm3 syringes
•10cm3 syringe
•stirring rod / •boiling tube
•test-tube rack
•stopwatch
•distilled water
•15cm3 of standard iodine solution
•60cm3 of stock starch suspension (10.0gdm–3) maintained at 25°C
•10cm3 of stock amylase solution (10.0gdm–3) maintained at 25°C
•eye protection
Introduction
In this practical, you will measure the course of the hydrolysis of starch by the enzyme amylase.
Iodine gives a blue-black colour with starch. The intensity of the colour is proportional to the concentration of the starch. This intensity can be measured in a colorimeter, and used to follow the course of the reaction. As the amylase hydrolyses the starch, the blue colour that starch forms with the iodine decreases in intensity.
Procedure
A Constructing a standard curve
1Using the measuring cylinders, dilute some standard iodine solution 1:20 with distilled water, in the large beaker. Make up 200cm3 of this diluted iodine. Stir to mix thoroughly.
2Set up the colorimeter with a red filter.
3Place some of the diluted iodine solution in a cuvette, and place the cuvette in the colorimeter.
Set the absorbance reading of the colorimeter to zero. This is the blank solution (i.e. one containing no starch). This blank must be kept for comparison throughout the rest of the experiment.
4The stock starch suspension has a concentration of 10.0gdm–3. Prepare ten dilutions of this stock solution as shown in the table below.
Stock / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10volume of stock starch suspension/ cm3 / 10.0 / 7.0 / 6.0 / 5.0 / 4.0 / 3.0 / 1.0 / 0.6 / 0.4 / 0.1 / 0.05
volume of distilled water/ cm3 / 0.0 / 3.0 / 4.0 / 5.0 / 6.0 / 7.0 / 9.0 / 9.4 / 9.6 / 9.9 / 9.95
concentration of diluted starch suspension/
g dm–3 / 10.0 / 7.0 / 6.0 / 5.0 / 4.0 / 3.0 / 1.0 / 0.6 / 0.4 / 0.1 / 0.05
5Place 5.0cm3 of the diluted iodine solution into a 25cm3 beaker. Use a 1cm3 syringe to add 0.5cm3 of the 10.0gdm–3 starch suspension to the iodine. Stir thoroughly. Pour this mixture into a second cuvette and obtain an absorbance reading in the colorimeter.
6Repeat step 5 for each of the starch suspensions you have prepared. Remember to wash out the syringe and beaker after you have tested each mixture.
7Draw a graph of absorbance against concentration of starch. This is a conversion graph, or standard curve.
B Following the course of the reaction
1Using a 5cm3syringe, place 5.0cm3 of the diluted iodine solution into a 25cm3 beaker.
2Place 15.0cm3 of starch suspension and 5.0cm3 of amylase solution from the stock in the water bath into separate plastic beakers, using the 10cm3syringe and a clean 5cm3syringe respectively. Mix these together and stir thoroughly. Now add 0.5cm3 of the mixture to the beaker of iodine solution, using the1cm3 syringe. Mix, and start the stopwatch. Pour some of the mixture into a cuvette, and take a colorimeter reading. This must be done quickly. Pour the remaining starch–amylase mixture into a boiling tube and place the mixture in the water bath.
3Wash the syringes and beaker with distilled water and place another 5.0cm3 of iodine solution into the beaker. Take another 0.5cm3 sample from the boiling tube 2minutes after the enzyme and substrate were first mixed, and repeat the procedure, sampling from the mixture every 2minutes for 30minutes (or as long as it takes to get a reasonable change in the colorimeter reading).
4Record the results in a suitable table. Use the standard curve to convert meter readings into concentrations of starch, and record these values in the table.
5Plot a graph of starch concentration against time.
6Describe the shape of the curve, and suggest an explanation to account for its shape.
COAS Biology 1 Teacher ResourcesOriginal material © Cambridge University Press 20081